Intercellular Transfer of Organelles in Plants for Horizontal Transfer of DNA Expressing Proteins of Interest

ABSTRACT

Compositions and methods for effecting horizontal gene transfer in plants are disclosed.

This application is a §365 Application of PCT/US11/68153 filed Dec. 30, 2011, which in turn claims priority to U.S. Provisional Application 61/428,672 filed Dec. 30, 2010, the entire disclosures of each being incorporated herein by reference as though set forth in full.

FIELD OF THE INVENTION

The present invention relates to plant genetic engineering and particularly to methods for horizontal gene transfer in higher plants.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Cells within a multicellular organism are connected by cytoplasmic bridges, which are termed plasmodesmata in plants (1) and tunneling nanotubes in animals (2). Plasmodesmata were shown to actively and passively regulate intercellular trafficking of viral proteins, transcription factors, phloem proteins, mRNA and sRNA in plants (1, 3). An important, recent development was the demonstration of the exchange of genetic material between cells in plant tissue grafts (4). However, there is no report yet on the intercellular movement of DNA-containing organelles, plastids and mitochondria, between plant cells.

During the past few years, supracellularity has emerged as a trait common to all life. Once thought to be a feature unique to plants, the physical continuity of cytoplasm and plasma membranes between neighboring cells has been observed in animal cells (2). These tunneling nanotubes were shown to be the conduits of active transport of organelles and cytoplasmic molecules between cells. Particularly relevant for this work, is the direct observation of transport of mitochondria through tunneling nanotubes in animal cells (5, 6). Tunneling nanotubes, filopodia-like cytoplasmic bridges have also been observed linking unrelated bacterial cells and therefore may represent a universal mechanism for cellular communication and interdependence (7).

Clearly, modulation of this process would represent an advance in the art in the creation of transplastomic plants.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for effecting intercellular transfer of organelles in plants for the creation of transgenic plants exhibiting desirable characteristics is provided. An exemplary method entails joining a root stock of a first plant and a scion from a second plant, said first and second plants comprising distinct plastid and nuclear genetic markers; culturing for a suitable period for grafting to occur; fragmenting or slicing the graft region and transferring said fragment or slices to a plant regeneration medium and selecting for cells expressing the nuclear and plastid genetic markers from said first and second plants. In one embodiment, the method entails decapitating the rootstock of a first plant, splitting the stem of said root stock and inserting a wedge shaped stem of scion from a second plant in the opening in the root stock, said first and second plants comprising distinct plastid and nuclear genetic markers; and culturing the graft plant for a suitable period for grafting to occur; then following the protocol above. The method can also comprise characterization of the size and type of DNA transferred. In a preferred embodiment, the organelle is a plastid and the method results in complete transfer of the plastid genome. In a particularly preferred aspect, the transferred plastid genome comprises at least one heterologous or endogenous DNA molecule expressing a protein of interest, e.g., a protein conferring herbicide or drought resistance. Other proteins of interest include without limitation, a fluorescent protein, an antibody, a cytokine, an interferon, a hormone, a selectable marker protein, a coagulation factor and/or an enzyme. Also provided are transgenic plants generated using the foregoing methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Phenotypes of the graft partners and the G1 graft transfer plant. (A) Partner P1 is N. tabacum (2N=48) with a nuclear gentamycin resistance transgene and wild-type N. tabacum plastids and mitochondria. Partner P2 has a wild-type N. sylvestris (2N=24) nuclear genome, N. undulata plastids with aadA transgenes for spectinomycin selection and the aurea young leaf color phenotype (bar^(au) gene) and N. undulata mitochondria that confer cytoplasmic male sterility (CMS-92). Shown is also the G1 plant and its markers. Black bar=10 cm (B) Flower morphology of the P1 and P2 partners and G1 PGT plant. White bar=1 cm

FIG. 2. Identification of plastid graft transfer events. (A) Grafted plant. Note that the P2 scion shown here is green because the expression of the bar^(au) gene is restricted to fast growing tissue and is sensitive to environmental conditions. (B) Selection in cultures of one- to two-mm graft sections for gentamycin- and spectinomycin-resistance. On the left are stem sections from above (P2) and below (P1) the graft and on the right from the graft region. Note a green, proliferating callus that yielded the G4 PGT plants.

FIG. 3. SSR markers confirm N. tabacum chromosomes in the G4 plant by testing each of the 24 chromosomes (numbered 1-24). Lanes are marked with s, G and t for the P2, G1 and P1 plants (see caption to FIG. 1). Some markers do not amplify the N. sylvestris template (8). White dots mark the 200-bp fragment of the 20-bp molecular weight ladder.

FIG. 4. Identification of the source of mtDNA in the PGT plants. (A) Schematic representation of the tobacco mtDNA master circle with the position of polymorphic regions marked. Repeated regions are marked with boxes. (B) Mitochondrial DNA sequence polymorphisms. (C) Map position of polymorphic sites relative to the sequencing primers and gene features.

FIG. 5. Identification of the N. undulata plastids in the PGT plants. (A) Identity plots of the plastid genomes of the transplastomic P2 partner carrying N. undulata ptDNA (u) with the aadA and bar^(au) transgenes (JN563930), the G1, G3, G4 (G) PGT plants and the P1 partner with N. tabacum ptDNA (t; Z00044) aligned with the mVISTA program using a 500-bp sliding window. Above the map are shown the positions of the DNA probes (#1 through #6) and DNA polymorphisms (*1 through *7). (B) Plastid DNA sequence polymorphisms. For map position see FIG. 5A. (C) DNA gel blot to identify RFLP markers in ptDNA. For probes see FIG. 5A.

FIG. 6. Model for cell-to-cell movement of plastids via initial cytoplasmic connection in graft junctions. (A) Cells at graft junction reconnect by plasmodesmata. Arrows point to sites where opposite parts of the contact walls are synchronously thinned (9). These are future sites of plasmodesmata. Proplastids (ovals), mitochondria (small circles) and nuclei (large circles) are identified in scion and rootstock. Ns, N. sylvestris; Nt, N. tabacum, Nu. N. undulata; CMS, cytoplasmic male sterile. (B) Proplastid is transferred via initial cytoplasmic connection. (C) Transferred spectinomycin resistant plastid takes over on selective medium. Note that the cells derive from the bottom cell in FIG. 6B.

DETAILED DESCRIPTION OF THE INVENTION

Land plants developed highly sophisticated intercellular channels or plasmodesmata, which mediate cell-to-cell movement of nutrients, hormones and information macromolecules. Functional equivalents of plasmodesmata in animal cells are the tunneling nanotubes, which were shown to mediate intercellular trafficking of organelles. Our objective was to test whether or not organelles move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new plasmodesmatal connections between the genetically distinct plants creating an opportunity for cell-to-cell organelle movement. We selected in tissue culture of the graft junctions for clonal lines, in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids.

Here we report cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in the form of intact plastids. We also report that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells via plasmodesmata provides a novel mechanism by which cells may be re-populated with functioning organelles. Our finding confirms the universal nature of intercellular organelle trafficking thus far documented only in mammalian cells and enables new biotechnological applications. Plastid transformation currently is a tissue culture dependent protocol that can be performed only with tissue-culture responsive genetic lines. Introduction of transformed plastid genomes into commercially useful lines requires repeated cycles of backcrosses. Inter-cellular transfer of organellar DNA in tissue grafts enables one-step transfer of plastid genomes in the absence of the transfer of nuclear genetic information, eliminating the need for backcrosses. Furthermore, graft transfer of plastidsis possible between sterile plants lacking flowers and between sexually incompatible genetic lines.

Inter-cellular transfer of plastid DNA is an example of horizontal gene transfer, defined as any process in which the recipient organism acquires genetic material from a donor organism by asexual means. Well studied is the role of massive horizontal gene transfer in the evolution of mitochondria and plastids from an alpha-proteobacterium and a cyanobacterium, respectively (Abdallah et al., 2000; Keeling and Palmer, 2008; Gross and Bhattacharya, 2009). Additional examples of horizontal gene transfer during evolution include prokaryote-eukaryote transfers, eukaryote-eukaryote transfers, eukaryote-prokaryote transfers and horizontal gene transfer in the plant cytoplasm (Richardson and Palmer, 2007; Keeling and Palmer, 2008; Bock, 2009).

The following definitions are provided to facilitate an understanding of the invention.

“Heteroplastomic” refers to the presence of a mixed population of different plastid genomes within a single plastid or in a population of plastids contained in plant cells or tissues.

“Homoplastomic” refers to a pure population of plastid genomes, either within a plastid or within a population contained in plant cells and tissues. Homoplastomic plastids, cells or tissues are genetically stable because they contain only one type of plastid genome. Hence, they remain homoplastomic even after the selection pressure has been removed, and selfed progeny are also homoplastomic. For purposes of the present invention, heteroplastomic populations of genomes that are functionally homoplastomic (i.e., contain only minor populations of wild-type DNA or transformed genomes with sequence variations) may be referred to herein as “functionally homoplastomic” or “substantially homoplastomic.” These types of cells or tissues can be readily purified to a homoplastomic state by continued selection.

“Plastome” refers to the genome of a plastid.

“Transplastome” refers to a transformed plastid genome.

“Transformation of plastids” refers to the stable integration of transforming DNA into the plastid genome that is transmitted to the seed progeny of plants containing the transformed plastids.

A “selectable marker gene” refers to a gene that upon expression confers a phenotype by which successfully transformed plastids or cells or tissues carrying the transformed plastid can be identified. Selectable marker genes as used herein can confer resistance to a selection agent in tissue culture and/or confer a phenotype which is identifiable upon visual inspection. Thus, in one embodiment the selectable marker gene can act as both the selection agent and the agent which enables visual identification of cells comprising transformed plastids. In an alternative embodiment, the selectable marker encoding nucleic acid comprises two sequences, one encoding a molecule that renders cells resistant to a selection agent in tissue culture and another that enables visual identification of cells comprising transformed plastids.

“Transforming DNA” refers to homologous DNA, or heterologous DNA flanked by homologous DNA, which when introduced into plastids becomes part of the plastid genome by homologous recombination.

“Agroinfiltration” refers to Agrobacterium mediated T-DNA transfer. Specifically, this process involves vacuum treatment of leaf segments in an Agrobacterium suspension and a subsequent release of vacuum, which facilitates entry of bacterium cells into the inter-cellular space.

“T-DNA” refers to the transferred-region of the Ti (tumor-inducing) plasmid of Agrobacterium tumefaciens. Ti plasmids are natural gene transfer systems for the introduction of heterologous nucleic acids into the nucleus of higher plants.

A “plant sector” refers to a region or a full leaf of a plant that is visually identifiable due to expression of a selectable marker gene or the excision of a selectable marker gene in accordance with the present invention.

“Operably linked” refers to two different regions or two separate genes spliced together in a construct such that both regions will function to promote gene expression and/or protein translation.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5′ to 3′ direction. With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

The terms “percent similarity”, “percent identity” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.

The term “functional” as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.

The phrase “consisting essentially of” when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

A “replicon” is any genetic element, for example, a plasmid, cosmid; bacmid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.

The term “oligonucleotide,” as used herein refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The term “probe” as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method.

The term “primer” as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.

All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.

The term “tag,” “tag sequence” or “protein tag” refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence.

The terms “transform”, “transfect”, “transduce”, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion, biolistic bombardment and the like.

A “clone” or “clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.

A “cell line” is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.

The following materials and methods are provided to facilitate the practice of the present invention.

Materials and Methods

Partner P1 (Nt-pHC19) has an allotetraploid Nicotiana tabacum cv. Petit Havana (2N=48) nucleus with the aacC1 transgene for gentamycin resistance and wild-type N. tabacum plastid and mitochondrial genomes (10). Partner P2 (Ns-pCK2-6W2) has a wild-type diploid N. sylvestris TW137 (2N=24) nuclear genome, N. undulata plastids with aadA transgenes for spectinomycin selection and the aurea young leaf color phenotype (bar^(au) gene), and cytoplasmic male sterile (CMS-92) mitochondria from N. undulata (11). For grafting, the plants were grown aseptically on a medium containing MS salts and 3% sucrose (12). Plants were regenerated from the graft junctions on RMOP shoot regeneration media supplemented with 500 mg/L spectinomycin and 100 mg/L gentamycin (12). Southern probing for ptDNA polymorphisms was carried out using six previously identified polymorphic regions (13). Organellar DNA was amplified using total cellular DNA as a template (14) using appropriate PCR primers (Table S1, Table S2). Primer design for ptDNA was based on GenBank Accession Z00044 and JN563929 and for mtDNA on GenBank Accession BA000042. The plastid genomes were amplified in 34 PCR reactions using primers listed in Table S3. DNA sequence was determined on an Illumina Genome Analyzer II using 80 bp paired-end (500 bp insert) library. Total leaf DNA fragments of P1, P2, G1, G3 and G4 plants were also analyzed on a SOLiD 5500×1 sequencer using 76-nucleotide reads. Reference guided assembly was essentially carried out as described (15). Nuclear SSR markers (8) were amplified using primers listed in Table S4.

TABLE S1 Plastid primers for testing ptDNA polymorphic sites between N. tabacum and N. undulata Pair Primer Position Strand Gene Sequence *1 12upF 12907 F atfP TCTTACTTAGAATAGGTCGTCGATTCAGCA *1 14upR 14098 R atfP CCACTGATTTCTGCCGCTTCCGTT *2 27upF 27875 F rpoB-trnC ACACATTCCAACCTGCTTGAATACCA *2 29upR 29210 R rpoB-trnC TCTTCCGCCCCCTTCCACAACTAT *3 48upF 48971 F trnL GAGACATTCCTCCGCTTTCAGGCG *3 49upR 49945 R trnL TGGAACCGCTAAGGAAAGGGGGTC *4 60upF 60806 F accD AACGGCATTCCCGTAGCAATTGGG *4 62upR 62222 R accD GGATGAGATTGGGTCCCAGCGGAT *5 83upF 83888 F ndhF TTTCCACCACGACGTGCATTTCGT *5 85upR 85414 R ndhF TACAAATTGCGGGGCGTATCGACG *6 111upF 111916 F ndhE-ndhG TCGGAAGAAAGGTGGGATCCGGAC *6 113upR 113293 R ndhE-ndhG TGGTATGGGGTCTTATCGAAGCGC

TABLE S2 Mitochondrial primers for testing mtDNA polymorphic sites between N. tabacum and N. undulata Pair Primer Position Strand Gene Sequence 1 mt-0-F 690 F orf125a CCCCGCCCAGTAGTGCCTCT 1 mt-4-R 4334 R orf125a CCGCGGGCATCGCGATAAGT 2 mt-100-F 100070 F orf129b CGGCCATCCTGGTCCTCAGGA 2 mt-104-R 104811 R orf129b TGGGGACTCGCACGAGGAGG 3 mt-180-F 180316 F nad4 GGCAGGAGCGCAACGACCTT 3 mt-183-R 183813 R nad4 AGTCGGGTTGCTCACGCAGC 4 mt-201-F 201586 F nad2 TGGTGTGCTTCCTGCTCGCG 4 mt-204-R 204759 R nad2 TTTCTCCGTGCCCGTTCCGC 5 mt-222-F 222140 F nad5 AGGTGCCCGTAGTAGGCCGG 5 mt-226-R 226463 R nad5 TTGGGCTTGGCTCTGCTCGC 6 mt-306-F 306203 F orf115-ccmFc CACGACTCCCCCTCTCCCCG 6 mt-309-R 309623 R orf115-ccmFc TGCCCGATTCCCCGACCCAT

TABLE S3 Plastid primers for PCR amplification of the Nicotiana  tabacum and N. undulata plastid genomes Pair Primer Position Strand Gene Sequence  1   0F     14 F trnH ACGGGAATTGAACCCGCGCA  1   4R   4410 R trnK CGGGTTGCTAACTCAACGG  2   3F   3704 F trnK TCAAATGATACATAGTGCGATACA  2   8R   8653 R trnS CGAATCCCTCTCTTTCCG  3   7F   7989 F psbK GCCTTTGTTTGGCAAGCTGCTGTAAG  3  12R  12042 R atpA GGCATTGCTCGTATTCACGGTCTTG  4  11F  11052 F atpA CCACTCTGGAAACGGAGATACCC  4  16R  16791 R rps2 CTCGTTTTTTATCAGAAGCTTGTG  5  15F  15267 F atpI GATGGCCCTCCATGGATTCACC  5  20R  20888 R rpoC2 GAGGATTAATGTCAGATCCTCAAGG  6  19F  19971 F rpoC2 GATAGACATCGGTACTCCAGTGC  6  24R  24612 R rpoB GTTACACAACAACCCCTTAGAGG  7  24F  24069 F rpoC1 GCACAAATTCCGCTTTTTATAGG  7  29R  29568 R ycf6 GCCCAAGCAAGACTTACTATATCCAT  8  28F  28849 F trnC CCAGTTCAAATCCGGGTGTC  8  34R  34493 R psbD TACCAAGGGCTATAGTCAT  9  33F  33186 F trnT GCCCTTTTAACTCAGTGGTA  9  38R  38115 R trnG AACCCGCATCTTCTCCTTGG 10  37F  37147 F trnS GAGAGAGAGGGATTCGAACC 10  43R  43484 R psaA TTCGTTCGCCGGAACCAGAA 11  41F  41267 F psaA AAGAATGCCCATGTTGTGGC 11  46R  46162 R ycf3 CCTATTACAGAGATGGTGCGATTT 12  45F  45083 F ycf3 CGATGCATATGTAGAAAGCC 12  51R  51022 R ndhJ TTTTTATGAAATACAAGATGCTC 13  49F  49312 F trnL CGAAATCGGTAGACGCTACG 13  54R  54971 R atpE GAAGGAAGGAGACAAAAAATTGAGGC 14  53F  53776 F trnV CGAACCGTAGACCTTCTCGG 14  58R  58198 R rbcL GTAAAATCAAGTCCACCGCG 15  57F  57272 F atpB TCTAGGATTTACATATACAACAT 15  62R  62754 R ycf4 CTAATAAGAAGCCTAATGAACC 16  61F  61145 F accD GCAGGTAAAAGAGTAATTGAAC 16  66R  66664 R psbL TACTCATTTTTGTACTTGCTGT 17  65F  65219 F petA GCATCTGTTATTTTGGCACA 17  71R  71704 R clpP ACCATAGAAACGAAGGAACCCACT 18  70F  70727 F rps18 GCTCGTATTTTATCTTTGTTACC 18  76R  76301 R psbB CCCCTTGGACTGCTACGAAAAACACC 19  74F  74963 F psbB TGCCTTGGTATCGTGTTCATAC 19  78R  78846 R petB CCCAGAAATACCTTGTTTACG 20  77F  77212 F psbH TGGGGAACTACTCCTTTGAT 20  82R  82676 R rps8 CGAGGTATAATGACAGACCGAG 21  81F  81880 F rp136 ATTCTACGTGCACCCTTACG 21  86R  86576 R rps19 GGGCATCTACCATTATACCC 22  85F  85864 F rps3 AGTCTGAAACCAAGTGGATTTATT 22  89R  89311 R YCF2 GAAGATACAGGAGCGAAACAATCAAC 23  88F  88062 F rp12 GCTTATGACCTCCCCCTCTATGC 23  93R  93140 R YCF2 TCTTCTAGAGAATCTCCTAATTGTTC 24  91F  91131 F YCF2 CTTCGAATATGGAATTCAAAGGGATC 24  97R  97636 R ndhB CTCAAACAAGCATGAAACGTATGC 25  96F  96469 f trnL GAGATTTTGAGTCTCGCGTGTC 25 100R 100782 R rps12 TCACTGCTTATATACCCGGTATTGGC 26  99F  99552 F rps7 GTGCAAAAGCTCTATTTGCCTCTGCC 26 104R 104797 R oriA ATCGAAAGTTGGATCTACATTGGATC 27 103F 103454 F rrn16 CGACACTGACACTGAGAGACGAAAGC 27 108R 108280 R rrn23 CGCTACCTTAGGACCGTTATAGTTAC 28 107F 107056 F rrn23 GAAACTAAGTGGAGGTCCGAACCGAC 28 111R 111882 R ORF350 AGTGGATCCCTCTTGTTCCTGTTTAG 29 110F 110672 F trnN ACAGCCGACCGCTCTACCACTGAGC 29 114R 114269 R ndhF GGATCATACCTTTCATTCCACTTCC 30 113F 113036 F ndhF ATTTCATCTTTGGACCAAAAACAAGC 30 119R 119286 R psaC GCTAAACAAATTGCTTCTGCTCC 31 117F 117227 F ycf5 GGTCAATCTTTTAGGAATAGGGTTAC 31 123R 123506 R ndhA GGACTTCTTATGTCGGGATATGGATC 32 122F 122194 F ndhA CTGCGCTTCCACTATATCAACTGTAC 32 128R 128835 R ycf1 TGAAACCTTGGCATATATCT 33 127F 127391 F ycf1 AATTTCGAGGTTCTTATTTACT 33 132R 132957 R trnR GACGATACTGTAGGGGAGGTC 34 154F  154629 F rp12 CCATAGAATACGACCCTAAT 34   1R   1533 R psbA CTAGCACTGAAAACCGTCTT

TABLE S4 Nuclear SSR Primers Chromosome Primer Strand Sequence  1 PT30307 F AAAGAAGCACGGTCAAATAGG  1 PT30307 R GCAACAACAAGGTGTCATGG  2 PT30242 F TGTGTACTACCGGCCTACTGC  2 PT30242 R TTCTGCTAAACCGATCGTGG  3b PT30205 F GGTCGATCCACAATTTAAACG  3b PT30205 R GCACTTGCTCCTTTGTACCC  4 PT30272 F GAACCTAACCTCGCTCCACA  4 PT30272 R AAATGGTAGCTGCGAGGAGA  5 PT30471 F GTCTGTACCTTCGCCAAAGC  5 PT30471 R TCCTCAGAGAACTCCAGCGT  6 PT30087 F CTTCTTCCTAAGCCGAGGGT  6 PT30087 R TTGATGATAGAACGCAACTCG  7 PT30138 F AGTTGCAGGATTGTTCGCTT  7 PT30138 R CGACTGCAAGAGTTGGCAAT  8a PT30167 F TGATACAGAATATGGCGAACTTT  8a PT30167 R CCGCTTCATCATTGAGGTTT  9 PT30140 F AAGATGGCATATGGGATTGG  9 PT30140 R TGAATCGGAGGAAGTGAATG 10 PT30482 F CTTCTCTCTCCACCGCAGAC 10 PT30482 R ACAGTTGGATATGGTGGCGT 11 PT30008 F CGTTGCTTAGTCTCGCACTG 11 PT30008 R GGTTGATCCGACACTATTACGA 12 PT30098 F TTGTTGCTCTCTCGAGTTCTTT 12 PT30098 R GCAGTCGACTCATTGGCA 13 PT30342 F GACAACAATCAGTAAAGGAAACGA 13 PT30342 R AATGCAAGACCCTGTCAACC 13 PT30420 F AACAAACCGCTTTCCATTCT 13 PT30420 R GAATTAGGCGCTTTGGGAAT 14a PT30175 F TTAGGCGGCGGTATTCTTAT 14a PT30175 R TATGCCTCAATCCCTTACGC 15 PT30463 F AAGCTGCCCTAGCTCAATCA 15 PT30463 R AACATCACCATTTCCACAAGTTT 16 PT30412 F CATTTAGCCGGGAACATTCA 16 PT30412 R CATGGGATACACACGCAAAG 17 PT30274 F TGACAGCTAAGCTAATAACAGTAAATG 17 PT30274 R GGACTTTGGAGTGTCAAATGC 18 PT30111 F AGCCAGCCACCAAATTTATC 18 PT30111 R GGAACATTGCTCAAGCCCTA 19 PT30230 F TTTCTTTCTGTCTGATGCTTCAAT 19 PT30230 R TTGTCCATCTCACTTGCTGC 20 PT20286 F ACGCTAGAGCATCCAACA 20 PT20286 R TAGTGAAAGGCAAGCAGG 21 PT30378 F TCAAATGAGGGTTGTAGCCA 21 PT30378 R TGCAATGGCTACACAAGAAGA 22 PT30168 F TTGAACACCAATTGCGGTAA 22 PT30168 R AAATTCTTGGGTCATGGTGG 23 PT30231 F AGGAGGCGAAGAAAGAGGAG 23 PT30231 R CCCATGAATTCGTAACAGCA 24 PT40024 F AATGTCTGCCCAATCGAAAG 24 PT40024 R CGAATAACGACACTCGAACG

EXAMPLE 1 Transmission of ptDNA in N. tabacum/N. sylvestris Graft Tissue

Our objective was to determine if chloroplasts or mitochondria could be shared among supracellular plant cells. To test this hypothesis, we grafted two different species of tobacco with genetic markers in their plastids and mitochondria. Grafting triggers formation of new plasmodesmatal connections (9) that creates a conduit for cell-to cell movement of organelles. We report here evidence supporting the transfer of plastids via newly formed plasmodesmata. However, the related (non-selected) mitochondria were absent in the same plants, suggesting independent transfer of plastids through the graft junction. We discuss acquisition of plastids from neighboring cells via plasmodesmata as a potential mechanism to repopulate cells with functional organelles and new opportunities created by the cell-to-cell movement of plastids for biotechnological applications.

Experimental Design.

Because of the difficulty to directly observe rare intercellular organelle movement, we chose graft partners with distinct nuclear and organellar genomes to test for cell-to-cell transfer of plastids and mitochondria in graft junctions (FIG. 1). We grafted two species of tobacco, Nicotiana tabacum (partner P1) with a selectable transgenic nuclear gentamycin resistance gene and Nicotiana sylvestris (partner P2) with plastids carrying a selectable spectinomycin resistance (aadA) gene and the aurea young leaf color phenotype (bar^(au) gene). The N. sylvestris partner carried the plastids and mitochondria of a third species, Nicotiana undulata, providing a large number of organellar DNA markers. The P1 partner with the N. tabacum nucleus was fertile and the P2 partner with the N. sylvestris nucleus cytoplasmic male sterile (CMS) (FIG. 1B), a trait controlled by mitochondria (16). The grafted plants were grown in culture for ten days (FIG. 2A) and sections of the graft junctions were selected for the gentamycin and spectinomycin resistance traits carried by the P1 nucleus and in P2 plastids, respectively (FIG. 2B). Out of 30 graft junctions a total of three plastid graft transmission (PGT) events (G1, G3, G4) were recovered. The plants regenerated from the graft junction displayed the leaf morphology, growth habit and pink flowers associated with the selected N. tabacum nucleus, but the aurea leaf color of the P2 partner, a plastid trait (FIGS. 1A and B).

No Exchange of Chromosomes in the PGT Plants.

To investigate the contribution of nuclear genetic material to the PGT plants, we examined twenty-four simple sequence repeat (SSR, or microsatellite) polymorphic DNA markers previously mapped to each of the N. tabacum chromosomes (8). These markers distinguished N. tabacum from N. sylvestris ecotype TW137 and indicated the presence of the chromosomes of the N. tabacum P1 partner that carried the selectable nuclear gene without contribution from the non-selected P2 N. sylvestris nucleus (FIG. 3). The presence of chromosomal markers from one partner excluded chimera formation as the source of double resistance of the G1, G3 and G4 PGT plants.

Mitochondria Remain Associated with the Selected Nucleus.

The graft partners carried distinct mitochondrial genomes determining the flower type (FIG. 1B). The P1 partner with the N. tabacum nucleus had normal anthers and produced fertile pollen while the P2 partner with the N. sylvestris nucleus had stigmatoid anthers, a phenotype controlled by mitochondria. Interestingly, the G1, G3 and G4 PGT plants were male fertile and lacked the stigmatoid anthers of the CMS P2 partner. In line with the flower morphology, the CMS92 mtDNA markers were absent in the G1, G3 and G4 plants. To determine the source of the mitochondrial genome in the PGT plants, we identified six SNP and indel markers that are suitable to distinguish the N. undulata CMS92 mtDNA (FIG. 4) from the fertile N. tabacum mtDNA (17). Sanger sequencing of PCR fragments indicated that the G1, G3 and G4 plants have the mitochondrial genome of the nuclear donor (FIG. 4). Thus, we did not find evidence for the transfer of mitochondrial DNA in the PGT plants. Given the tendency of mitochondria for fusion (18) and mtDNA for recombination (16, 19), would mtDNA be transferred, we would expect to find at least chimeric mtDNA. The absence of non-selected mitochondrial DNA suggests limited organelle transfer that did not involve large-scale mixing of the two cytoplasms at the graft junction. Although we did not find evidence for the co-transfer of mtDNA transfer with ptDNA in the lines tested, it is possible that mtDNA transfer could be detected in a larger PGT plant population.

PGT Plants Contain the Entire Selected Plastid Genome.

Dual selection for the nucleus- and plastid-encoded antibiotic resistances ensured that the PGT plants would carry both transgenes. The N. tabacum-specific SSR markers in the G1, G3 and G4 plants indicated the presence of the P1 chromosomes alone in the PGT plants. However, the presence of the plastid markers did not distinguish between a transformation-like process that involves incorporation of ptDNA fragments and intercellular movement of plastids implied by the transfer of complete plastid genomes, either of which is compatible with the earlier report (4). To determine how much of the P2 ptDNA is present in the G1, G3 and G4 plants, we first examined markers distant from the transgenes by probing total cellular DNA on blots. Southern probing of the six previously identified RFLP markers (FIG. 5C) and PCR analyses (FIG. 5B) suggested the presence of the entire plastid genome of P2 partner and that the PGT plants carried a uniform population of P2 transplastomes. To exhaust the search for a contribution to the G1 plastid genome from the non-selected P1 plastome, we performed next generation sequencing of the plastid genomes of the P1 and P2 partners and the G1, G3 and G4 PGT plants. We report here the sequence of the 160,743 nucleotide transplastomes in the P2 partner and the three PGT plants are identical (GenBank accession no. JN563930). The P2 and PGT plastid genomes are larger than the 155,863 nucleotide wild type N. undulata plastid genome (GenBank accession no. JN563929), as the transplastomes also contain spectinomycin resistance (aadA) and the aurea bar^(au) transgenes. We also sequenced the plastid genome in partner P1 that carries the wild-type N. tabacum ptDNA of cv. Petit Havana. We have found that the sequence of cv. Petit Havana ptDNA is identical with the cv. Bright Yellow sequence deposited in GenBank (GenBank Accession number Z00044). However, the N. undulata ptDNA differs from the N. tabacum cv. Petit Havana ptDNA by 805 SNPs, 52 insertions and 61 deletions and the transgene cassettes. Differences between the plastid genomes are depicted on the mVISTA identity plots shown in FIG. 5A. Importantly, we observed all of these polymorphic loci, with an average density of 200 bp/SNP (170 bp/polymorphism) in the PGT graft transmission plants indicating the transfer of intact ptDNA from the P2 graft partner. We also tested transmission of the plastid-encoded spectinomycin resistance in reciprocal backcrosses. When the G1 plant was the mother and the wild type the father, each of the 208 seedlings was resistant whereas when the G1 plant was the father and the wild type the mother, each of the 318 seedlings was spectinomycin sensitive. Thus, spectinomycin resistance exhibited uniform, maternal inheritance, as expected for a homoplastomic N. tabacum, a species with strict maternal plastid inheritance.

Cell-to-Cell Migration of Plastids.

We report here cell-to-cell movement of entire plastid genomes. We considered two possible mechanisms for the transfer of genome-size ptDNA: the intercellular transport of extra-organellar (“naked”) DNA or the ptDNA traveling within an intact organelle. Selection for movement of ptDNA to the nucleus lead to the discovery of ptDNA transfer to the nucleus by incorporation of kilobase-size ptDNA fragments, most probably from degraded organellar genomes (20-22). Movement of entire genomes may require more protection than the fragments. Better protection could be provided if the extra-organellar ptDNA would be encapsulated in membrane-bound vesicles that are shed from fragmented chloroplast stromules (23). Because of the need for capacity for translation, plastids cannot be created de novo from membranes and DNA (24). Thus, if “naked” ptDNA is transferred, an invading plastome would need to enter an existing plastid with transcription and translation machinery and displace the existing plastome by a transformation-like process to explain our observations. However, a transformation-like process would yield mosaic genomes if different genomes were present, because plastid genomes within an organelle undergo frequent recombination (25-27). The absence of chimeric genomes in the PGT plants makes it unlikely that naked DNA transfer is the mechanism of intercellular ptDNA transfer.

More likely vehicles of cell-to-cell movement of entire plastid genomes could be the organelles themselves. The avenue for the movement of intact organelles could be damage to cell walls that allows for some mixing of cytoplasms in the graft junctions. A more likely mechanism would be the transfer of proplastids via newly formed connections between cells that are well documented at graft junctions (9). The size of proplastids, about one micrometer, is well above the size exclusion limit of plasmodesmata normally defined by molecular weight. However, the size exclusion limit changes during development and depends on tissue type (1, 28). We speculate that the new openings, formed by thinning of opposing cell walls at the site of future plasmodesmata, permit intercellular movement of proplastids. Our preferred model of intercellular plastid transfer in graft junctions is shown in FIG. 6.

The capacity of a plant cell to acquire organelles from a neighboring cell is a basic biological process. Acquisition of plastids from neighboring cells may be important because once the ribosomes are lost, translation cannot be restored, since some of the ribosomal proteins are encoded in the plastid genome and their translation is dependent on plastid ribosomes (24). Therefore, during certain stages of development, including dedifferentiation associated with forming new connections in grafted tissues (9), the plasmodesmata may allow the transport of organelles to ensure the continuity of functional DNA containing organelles. In this regard it is intriguing to note that the redox state of plastids regulates symplastic permeability and that ectopic expression of the proplastid-targeted GAT1 protein increased plasmodesmal size exclusion limit (29). The functional state of mitochondria also regulates the size exclusion limit of intercellular trafficking (30) and reprogramming of diseased mammalian cells was associated with acquisition of functional mitochondria (6). The discovery of intercellular movement of plastids now enables testing the biological significance of this process in plants.

While the protocol described here is based on wedge grafting, decapitation of the rootstock and separation of scion from its root system is not necessary to obtain grafting. Natural grafting has been observed between plants in nature, when graft junction forms between plants growing in close proximity (31). Accordingly, wedge grafting may be replaced by alternative protocols based on natural grafting. In one approach, the surface of the stem of the graft partners are removed and the stems are tied together to mimic natural grafts. PGT plants can be recovered from the graft junctions by tissue culture selection as described in the present application, or identified based on plant morphological markers and visual plastid markers in shoots regenerated from the graft junction. See U.S. patent application Ser. No. 13/326,295.

Intercellular movement of organelles should not be limited to intact plants, but should be applicable to any two cells making a new contact enabling cell-to-cell movement of plant organelles. Such cells may be in tissue culture, said first and second plants comprising distinct plastid and nuclear genetic markers, enabling selection for PGT events. Recovery of PGT (organelle) events in tissue culture may be particularly beneficial when grafting is technically challenging, such as in monocotyledonous plants.

Applications in Plastid Genetics and Biotechnology.

Because in most species both plastids and mitochondria are maternally inherited, they cannot be separated by crossing. Thus far protoplast fusion has been the only option to obtain new combinations of plastids and mitochondria (16). The result is intercellular transfer of parental plastids, but formation of recombinant mitochondrial genomes. The protocol we report here enables combination of parental plastids and non-recombinant mitochondria by PGT, a significant improvement over the protoplast-based process that yields recombinant mitochondria.

An additional application of PGT could be rapid introgression of transformed plastids into commercial cultivars. Plastid transformation is a powerful tool for biotechnological applications because the transgenes that are integrated into the plastid genome are expressed at high levels, can be clustered in operons and are not subject to silencing (32, 33). Currently the option is to transform the plastids in permissive cultivars then introduce them into commercial lines by repeated backcrossing using the commercial cultivar as a recurrent pollen parent. Based on the findings disclosed herein, backcrossing can be replaced in the future by graft transfer of the transformed plastids, instantly yielding a substitution line carrying the valuable commercial nuclear genome combined with transgenic plastids.

EXAMPLE 2 Introduction of Autoluminescent Chloroplasts into Genetically Sterile Plants or into Plants Lacking Flowers

Plastid transformation currently is a tissue culture dependent protocol that can be performed only with tissue-culture responsive genetic lines. Introduction of transformed plastid genomes into commercially useful lines requires repeated cycles of backcrosses. Inter-cellular transfer of organellar DNA in tissue grafts enables one-step transfer of plastid genomes in the absence of the transfer of nuclear genetic information, eliminating the need for backcrosses. Furthermore, graft transfer of plastids is possible between sterile plants lacking flowers and between sexually incompatible genetic lines.

Desirable plastids for transfer by non-sexual means may be autoluminescent plastids of different plant species carrying the lux operon (34) and the following recipients:

-   (1) Fertile lines that are sexually compatible, but encode desirable     traits in their nuclei. -   (2) Fertile lines that are sexually incompatible, thus introduction     could not be accomplished by crossing. -   (3) Plants, which lack flower organs or have flower organs but are     sterile.

EXAMPLE 3 Transmission of mtDNA in the N. tabacum/N. sylvestris Graft

We did not find evidence for co-transfer of the non-selected mtDNA with the selected ptDNA. Even if the mitochondria (mtDNA) were co-transferred with plastids, they were likely lost due to the absence of direct selection for mitochondrial traits. Thus, testing a larger population of PGT plants could possibly yield plants expressing the CMS flower morphology, a mitochondrial trait. A factor in the lack of recovering CMS plants could be the presumed recessive nature of Nicotiana undulata CMS, implied by the relatively small number of CMS plants recovered in somatic hybrids (35). Because in our case plastids from the CMS P2 partner have moved into the fertile P1 partner, if recessive, the CMS mitochondrial trait remains undetected, unless the dominant fertile mitochondrial determinants are lost. In order to increase the likelihood of detecting the co-transfer of mitochondria (mtDNA) with plastids, we will utilize fertile plants as the source of plastids, because detecting restoration of fertile flower morphology, a dominant trait, in a sterile partner is more likely in regenerated plants.

It is clear that the foregoing methods are useful for engineering plants and crops having desirable characteristics without the need for extensive back crossing.

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While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims. 

What is claimed is:
 1. A method for effecting intercellular transfer of organelles in plants for the creation of transgenic plants exhibiting desirable characteristics comprising: a) joining the cells of the first plant and a second plant, said first and second plants comprising distinct plastid and nuclear genetic markers; or b) joining a first plant and a second plant, said first and second plants comprising distinct plastid and nuclear genetic markers; or c) joining a root stock of a first plant and a scion from a second plant, said first and second plants comprising distinct plastid and nuclear genetic markers; and d) culturing said plants for a suitable period for grafting to occur; i) fragmenting or slicing the graft region and ii) transferring said fragment or slice to a plant regeneration medium and selecting for cells expressing the nuclear and plastid genetic markers from said first and second plants; or, e) forcing shoot formation from the graft junction and i) identifying plastid gene transfer events by altered plant morphology and/or visually detectable plastid-specific markers.
 2. The method of claim 1, further comprising characterization of the size and type of DNA transferred.
 3. The method of claim 1, wherein said organelle is a plastid and said method results in complete transfer of the plastid genome.
 4. The method of claim 3, wherein said transferred plastid genome comprises at least one heterologous DNA molecule expressing at least one protein of interest.
 5. The method of claim 4, wherein said heterologous DNA encodes a protein conferring herbicide or drought resistance.
 6. The method of claim 4, wherein said heterologous DNA encodes a protein selected from the group consisting of a fluorescent protein, an antibody, a cytokine, an interferon, a hormone, a selectable marker protein, a coagulation factor and an enzyme or a gene for a small RNA.
 7. The method of claim 1, wherein said plant is a solanaceous plant.
 8. The method of claim 1, wherein said plant is selected from the group consisting of Nicotiana tabacum, Nicotiana sylvestris, Nicotiana benthamiana, potato, tomato, and eggplant.
 9. The method of claim 1, wherein said plant is a dicotyledonous plant.
 10. A plant regenerated from the cells from step c) of claim
 1. 11. Progeny and seed from the plant of claim
 10. 